The long-term objective of this project is to develop multi-modal (MRI, fluorescence) 129Xe biosensors for early cancer detection, and possible therapeutic intervention. Cryptophanes bind xenon more tightly within their interior (Kd equals approximately 100 mu m) than other organic molecules, making these cages attractive for xenon imaging experiments. Xenon gas is sensitive to its molecular environment, and small perturbations near the cryptophane produce large changes in the 129Xe NMR chemical shift. Moreover, xenon is biologically compatible, diffuses readily in vivo, and can be "hyperpolarized" to enhance the NMR signal 10,000-fold. These properties form the basis for a sensitive imaging reagent capable of simultaneously monitoring multiple analytes in vivo, each causing a unique 129Xe chemical shift. This "multiplexing" capability offers unparalleled opportunities for molecular imaging of tumorigenesis, where simultaneous identification of multiple cancer markers would speed diagnosis, and improve treatment. Two particularly challenging, but important goals are the early detection of pancreatic and brain cancers, as these neoplasms are a leading cause of cancer death in the U.S., and their regulation is poorly understood at the molecular level. In the R21 portion of this proposal, Aim #1 is to synthesize novel cryptophanes with enhanced biosensing capabilities, including higher affinity for 129Xe, longer T1, and new chemical shifts. 129Xe*cryptophane binding will be characterized by NMR studies. Aim #2 is to functionalize cryptophane-A, currently the cage with highest affinity for xenon, with a variety of fluorophores for bimodal detection, as well as targeting agents such as cyclopamine, folate, glucose, and somatostatin. Enhanced cryptophanes developed in Aim #1 will also be functionalized. Aim #3 is to demonstrate specific delivery in tissue culture. Cryptophanes will be synthesized for optimal cell delivery and specificity with minimal toxicity. In the R33 phase, Aim #4 is to detect multiple markers simultaneously in cancer cell lines, using xenon delivery agents optimized for specific targets. Data collection procedures will be tested for in vivo applications: These include optimizing 129Xe NMR imaging parameters, protocols for delivering cryptophanes and hyperpolarized xenon, and streamlining data analysis. By this stage, an "arsenal" of cryptophanes will be in hand, and their toxicity will be tested in mice. Finally, Aim #5 is to demonstrate the early detection of pancreatic and/or brain tumors in mice using MRI. Biosensor deliverability, tumor selectivity, marker specificity, and 129Xe signal intensity will be evaluated in mouse tumor models. 129Xe MRI results will be validated using fluorescently labeled carriers and subsequently imaging tissue in vivo and ex vivo: such "ground truthing" will help to evaluate the diagnostic accuracy of this new technology. This proposal will focus on developing 129Xe reagents for the in vivo detection of multiple biomarkers associated with pancreatic and brain cancers.